Modular promoter construct

ABSTRACT

A modular promoter construct is described which possesses a promoter which is active in plant cells and a DNA sequence from exon 1 of the rice actin 1 gene. This modular promoter construct gives rise, where appropriate together with additional regulatory DNA sequences, to a substantial increase in gene expression in plant cells.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The invention relates to a modular promoter construct based on exon 1 ofthe actin 1 gene from rice.

There has always been the need to provide agronomically important cerealplants with improved properties. Previously, hereditary traits or geneswere crossed into the plants concerned using classical methods. Sincerecombinant DNA technology has become established, it is possible toinsert defined genes into the genome of cereal plants such as, forexample, rice, maize, wheat and barley. In addition to selecting thecorrect selective marker gene and the agronomically important genes, thechoice of the correct promoter is of crucial importance for effectivelyexpressing the desired gene. The promoter which is used most forincreasing the expression of chimeric genes in monocotyledonous cerealplants derives from the cauliflower mosaic virus (CaMV) 35S RNA gene(Odell et al., Nature 313, pages 810 to 820, 1985). However, as comparedwith its activity in dicotyledonous plants, this promoter is not veryactive in monocotyledonous plants (Topfer et al., Meth. Enzymol., "Rec.DNA", in press). Thus, alternative gene expression vectors using strongpromoters, such as the promoters of the actin 1 gene from rice (McElroyet al., Plant Cell 2, pages 163 to 171, 1990) and the ubiquitin genefrom maize (Christensen and Fox, International Society for PlantMolecular Biology Meeting, Program Abstracts, No. 287, 1991).

In analogy with animal systems (Dynan, Cell 58, pages 1 to 4, 1989), itis probably also the case for plant genes as well that the transcriptionmediated by RNA polymerase II is dependent on modular elements, i.e.elements consisting of different regulatory DNA sequences. It hasalready been demonstrated that a number of different modular elementsplay an important role in, for example, tissue-specific transcription(Katagiri and Chua, Trends Gent. 8, pages 22 to 70, 1992).

Thus, DE-OS 41 24 537 discloses a modular promoter construction whichcan be used to increase the expression of foreign genes in plant cells.In this case, a DNA sequence from exon 1 of the sucrose synthase genefrom Zea mays L. is inserted between the promoter and the gene to beexpressed. A further, multiplicative increase in gene expression ispossible if an additional DNA sequence, corresponding essentially tointron 1 of the sucrose synthase gene from Zea mays L., is coupled tothe said DNA sequence.

McElroy et al., loc. cit., have described the isolation of promotersequences of the rice actin 1 gene lying in the 5' region. It hasemerged that there is a positive correlation between the promotersequences and the subsequent intron sequences. Thus, in investigationsof the transformation of rice protoplasts, a promoter construct of thesesequences has proved to be an effective regulator of the constitutiveexpression of a foreign gene.

SUMMARY OF THE INVENTION

The underlying object of the present invention is to make available apromoter which can be used to express foreign genes in plants with ahigh degree of efficiency.

This object is achieved by a modular promoter construct according toPatent claim 1.

The subclaims relate to preferred embodiments of the novel promoterconstruct.

The invention thus relates to a modular promoter construct which has apromoter which is active in plant cells and a DNA sequence from exon 1of the rice actin 1 gene, and to the alleles and derivatives of thismodular promoter construct.

The invention furthermore relates to vectors which contain the novelpromoter constructs.

The invention also relates to plant cells which are transformed with thesaid vectors.

The invention additionally relates to plants, and their descendants,which are regenerated from the said plant cells.

Finally, the invention also relates to the use of the novel promoterconstructs for preparing plants having elevated gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures serve to elucidate the invention:

FIG. 1 shows chimeric gene constructs for transient gene expressionexperiments,

FIG. 2 shows a schematic representation of the rice actin 1 (act 1) genewith the exon 1 DNA sequence (SEQ ID NO:1) employed,

FIG. 3 shows a thin layer chromatogram of the transient expression ofchimeric constructs in Hordeum vulgare protoplasts,

FIG. 4 shows a thin layer chromatogram of the transient expression ofchimeric constructs in Triticum monococcum protoplasts, and

FIG. 5 shows a thin layer chromatogram of further transient geneexpression in Hordeum vulgare protoplasts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The novel promoter construct is a promoter which stimulates RNApolymerase II. The novel modular promoter construct possesses a promoterwhich is active in plant cells and a regulatory DNA sequence coupled tothis promoter. This DNA sequence derives from exon 1 of the actin 1 gene(act1/exon1 sequence) from rice. The DNA sequence contained in thepromoter construct preferably conforms to the sequence from positions +4to +57 in exon 1 of the actin 1 gene, as shown in FIG. 2 (SEQ ID NO:1).The untranslated exon 1 is separated by an intron (intron 1) from thetranslation start point in exon 2. Exon 1 is thus situated in the 5'region of the actin 1 gene which is transcribed but not translated. Theentire sequence of exon 1 of the said gene is given in McElroy et al.,(loc. cit.).

The act1/exon1 sequence is GC-rich (77%) and acts as an RNA-polymeraseII-stimulating element when it is located downstream of thetranscription start site.

It has been found that when the novel modular promoter construct iscoupled- to a gene which is to be expressed in a plant cell, theexpression of this gene is then substantially increased, it beingpossible to observe at least a 10-fold increase in expression of thegene.

The expression of the gene can be elevated a further 100-fold if theact1/exon1 sequence is combined with intron 1 of the Zea mays sucrosesynthase gene, surprisingly resulting in at least a 1000-fold increasein the expression of the gene.

It has emerged that the 18-bp OTF binding site of the octopine synthasepromoter (OCS enhancer) is compatible for example, with the CaMV 35Spromoter. If the OTF binding site is combined with the act1/exon1sequence and intron 1 of the Zea mays L. sucrose synthase gene, theactivity of the gene increases sharply by at least a factor of 4000.

It is evident from the above that gene expression can be multiplieddepending on the regulatory DNA sequence which is coupled to the gene tobe expressed. Thus, the novel modular promoter construct can be used toachieve a multiplication in gene activity.

The novel modular promoter constructs, and the vectors, contain, as apromoter which is active in plant cells, such as, for example, the CaMV35S promoter, the nopaline synthase promoter or the sucrose synthasepromoter.

The novel modular promoter construct is suitable for expressing alltypes of foreign genes, such as, for example, resistance genes, asdisclosed, for example, in EP-A 0 257 542 and EP-A 0 275 957, and alsofor preparing proteinogenous active compounds in plants, for example inaccordance with DD-A 12 65 164. Genes for storage proteins, such as, forexample, zeins or hordeins, are of particular importance.

Downstream of the gene to be expressed, the novel modular promoterconstructs, and the vectors which contain the novel promoter constructs,contain polyadenylation regions from the CaMV 35S gene or the octopinesynthase (OCS) gene (Hein et al., Mol. Gen. Genet., 199, pages 161 to168 (1985)).

The novel modular promoter construct is suitable for expressing foreigngenes in monocotyledonous and dicotyledonous plants. Examples ofsuitable monocotyledonous plants are cereal plants, such as wheat,barley, maize and rice. Tobacco (Nicotiana tabaccum) is an example of adicotyledonous plant which can be transformed satisfactorily.

It is self-evident that allelic variants and derivatives of theabovementioned modular promoter construct are also included within thescope of the invention, provided that these modified modular promoterconstructs perform the same function as the above-mentioned promoterconstructs, i.e. have a stimulatory effect on gene expression. Theallelic variations and derivatives include, for example, deletions,substitutions, insertions, inversions or additions of the individualsequences of the novel promoter construct.

The transformation of plants with a foreign gene using the novel modularpromoter construct can be carried out with the aid of customarytransformation methods. That which is particularly preferred isprotoplast transformation, followed by regeneration into a plant of theplant cells resulting from the transformed protoplasts.

Embodiments of the novel modular promoter construct which areparticularly preferred are described in the following examples.

EXAMPLE 1 Plasmid Constructions

The plasmid constructions described below are given in FIG. 1. Thechimeric gene constructions shown in this figure were used for transientgene expression experiments. The transcription start site and the CaMV35S gene polyadenylation signal are the same in all constructions. Therestriction sites which are of relevance to the cloning have also beenincluded. The polylinker (P) between the transcription start and thetranslation start encompasses, from the 5' end to the 3' end, therestriction sites X, D, EI and K. The restriction sites are abbreviatedas follows: BclI (B), EcoRI (EI), EcoRV (EV), HindIII (H), HincII (A),KpnI (K), SmaI (S), SstII (C) and XhoI (X). The CaMV 35S promoter isdenoted by "enhancer core" and the OTF binding site by "OCS".

All the plasmid constructions were prepared by customary methods, asdescribed, for example, in Sambrook et al., Molecular cloning: Alaboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989.

All the plasmid constructions are derived from the chimeric plasmid pRT101 CAT ab (Prols et al., Plant Cell. Rep. 7, pages 221 to 224, 1988),which contains the CAT marker gene (CAT=chloramphenicol transacetylase).In order to prepare the chimeric plasmid pCM 1106, the non-translatedsequence of exon 1 of the actin 1 gene (act1/exon1 sequence) frompositions +4 to +57 (SEQ ID No:1) was inserted into the unique SmaIrestriction site (S) in the 5'-non-translated leader of the pRT 101 CATplasmid. In this context, reference may be made to FIG. 2, which showsthe sequence from exon 1 which is employed and its position withinexon 1. Insertion of the said DNA sequence restores the SmaI restrictionsite due to 3'-terminal cytosine residues of the actin I element (seealso FIG. 2, positions +55 to +57).

The intron I sequences from the maize sucrose synthase gene (Sh1)(positions +43 to +1084) are isolated as a HincII restriction fragmentfrom the chimeric plasmid pSP 1076+1084, which is disclosed in DE-OS 4124 537, and this fragment is inserted into the SmaI restriction site ofpCM 1106. In this way, the chimeric plasmid construction pCM 1111 isobtained.

A DNA Synthesizer (Applied Biosystems 380 B) is used to synthesize thesequence from exon 1 of the rice actin 1 gene, shown in FIG. 2, and the18-bp OTF binding site having the sequence AACGTAAGCGCTTACGTT (Ellis etal., EMBO J. 6, pages 3203 to 3208, 1987). The OTF binding site is thensubcloned into the unique Smal restriction site in the commerciallyavailable plasmid pUC 19, resulting in pOTF 18. In order to prepare thechimeric plasmids pCM 1112 and pCM 2107, a HincII (A)/HindIII (H)restriction fragment is isolated from pCM 1111 and pCM 2106 (HindIII isa partial digestion of pCM 1111) and inserted into the unique HincIIrestriction site in pOTF 18.

In order to prepare the plasmid pCM 1107, the polylinker in the chimericplasmid pCM 1106 is removed by a restriction digestion using XhoI(X)/KpnI (K). The protruding ends are removed by careful treatment withS1. Autoligation results in the plasmid construct pCM 1107, which, incomparison to pCM 1106, has a deletion of 11 base pairs between thetranscription start site and the act1/exon1 sequence.

The promoter deletion plasmid pCM 2106 is prepared by removing CaMV 35Spromoter sequences from pCM 1106 upstream of the EcoRV (EV) restrictionsite at position -90.

EXAMPLE 2 2.1 Analysis of Transient Expression

Protoplasts are isolated from a cell suspension of the barley lineHordeum vulgare L. cv. Golden Promise in accordance with the methoddescribed for maize (Maas and Werr, Plant Cell. Rep. 8, pages 148 to151, 1989), using an osmolarity of 720 mosm. A cell suspension of theline Triticum monoccum (one-grained wheat) was cultivated, andprotoplasts were isolated, essentially as described by Lorz et al., Mol.Gen. Genet. 199, pages 178 to 182, 1985 and Matzeit et al., Plant Cell3, pages 247 to 258, 1991).

The Hordeum vulgare and Triticum monoccum protoplasts were transformedas described in Maas and Werr loc. cit. and Maas et al., loc. cit.Approximately 1 x 10⁶ protoplasts are transformed with 25 μg of plasmidDNA and 100 μg of sonicated calf thymus DNA, and a PEG-mediated genetransfer is carried out (PEG solution: 25% PEG (1500), 0.1 M MgCl₂, pH6.0). Gene expression is examined on the basis of the content of proteinformed at from 15 to 19 hours after the transformation.

2.2 Determination of CAT Activity

For the determination of CAT activity, the transformed cells arecentrifuged down, resuspended in 60 μl of 500 mM Tris/HCl at a pH of 7.5(2 mM PMSF) and lysed by two cycles of freezing and thawing. The extractis clarified by centrifugation. 20 μl of the supernatant are used fordetermining protein in accordance with Bradford, Anal. Biochem. 72,pages 248 to 254, 1976. CAT activity is determined using thin-layerchromatography essentially as described by Gorman et al., Mol. Cell.Biol. 2, pages 1044 to 1051, 1982. The extracts were preincubated at 65°C. for 10 minutes, with 250 μg of BSA being added to the reactionmixture.

Two transformations were carried out in parallel for each plasmidconstruct. The treated protoplasts are cultured and combined in order toreduce the differences due to the different transformation efficiencies.One half (one×10⁶ protoplasts) of this mixture is analysed for CATactivity. Owing to the high CAT activities in undiluted extracts, aseries of 1:10 dilutions (1:10, 1:100, 1:1000) is prepared in orderthereby to obtain linear CAT activities for the densitometricexamination of the autoradiographs.

2.3 Investigation Results

2.3.1 Hordeum vulgare

The investigations of gene expression in Hordeum vulgare protoplastscontaining the chimeric plasmids described in Example 1 led to thefollowing results.

The results for the relative CAT activities can be seen from thethin-layer chromatogram in FIG. 3. Due to the high CAT activities in thecrude extract, the activity was measured in a 1:100 dilution containing1×10⁴ protoplasts. The abbreviations have the following meanings:CAT=chloramphenicol transacetylase; Cm=C¹⁴ -labelled chloramphenicolalone; cont=protein extracts of non-transformed control protoplasts, and1, 3, and 1, 3 Cm=chloramphenicol acetylated at the given positions.

There is at least a 10-fold increase in marker gene expression in thecase of the chimeric plasmid pCM 1106 as compared with the referenceplasmid pRT 101 CAT.

It can be seen from the chimeric plasmid pCM 1111 that marker geneexpression is increased at least 1000-fold when the act1/exon1 sequenceis combined with the intron 1 sequences from the sucrose synthase gene.

The 18-bp OTF binding site of the octopine synthase promoter, togetherwith the entire CaMV 35S promoter, can interact with the act1/exon1sequence and intron 1 of the sucrose synthase gene. Insertion of the OTFbinding site into the chimeric plasmid pCM 1111, which contains theact1/exon1 sequence and sequences from intron 1 of the sucrose synthasegene, results in at least a further 3- to 4-fold increase in thestimulation ratio (see pCM 1112). This result is also obtained using a1:1000-fold dilution (not shown). Thus, it is demonstrated unequivocallythat the stimulatory effect of combining different individual DNAsequences is multiplicative and not additive.

In this connection, reference is made to Table 1 below, which lists theCAT activities, from FIG. 3, of the chimeric plasmids pCM 1106, pCM 1111and pCM 1112 in Hordeum vulgare protoplasts in relation to that of thereference plasmid pRT 101 CAT (activity=1).

                  TABLE 1    ______________________________________    pRT 101 CAT pCM 1106   pCM 1111 pCM 1112    ______________________________________    1           7.5-12.5   872-1242 3897-6965    ______________________________________

2.3.2. Triticum monoccum

The investigations of gene expression in Triticum monococcum protoplastscontaining the chimeric plasmids described in Example 1 led to thefollowing results.

As can be seen from FIG. 4, the same gene-expression stimulation ratiosare obtained with transformed formed Triticum monoccum protoplasts asare obtained with Hordeum vulgare protoplasts. Thus, insertion of theact1/exon1 sequence results in at least a 10-fold increase in markergene expression (pCM 1106). Combination of the act1/exon1 sequence withthe intron 1 sequences of the sucrose synthase gene results in at leasta 1000-fold increase in gene expression.

In this connection, reference may be made to Table 2 below, in which theCAT activities of the chimeric plasmids pCM 1106 and pCM 1111 from FIG.4 are related to that of the chimeric reference plasmid pRT 101 CAT(activity=1).

                  TABLE 2    ______________________________________    pRT 101 CAT     pCM 1106 pCM 1111    ______________________________________    1               7.5-11.9 913-1203    ______________________________________

The thin-layer chromatogram in FIG. 5 demonstrates that the act1/exon1sequence does not give rise to any increase in gene expression withoutthe presence of upstream regulatory sequences. The act1/exon1 sequenceincreases marker gene expression at least 10-fold when it is inserteddownstream of the entire CaMV 35S promoter (pCM 1106). By contrast,deletion of the CaMV 35S promoter in the chimeric plasmid pCM 1106 (pCM2106, FIG. 1) completely abolishes the stimulatory ability of theact1/exon1 sequence.

Since, for effective marker gene expression, the act1/exon1 sequencerequires the presence of upstream regulatory elements, the activity ofthe chimeric plasmid pCM 2106 can be restored by inserting upstreamregulatory elements. For this reason, the 18-bp OTF binding site wasinserted, as a regulatory sequence, upstream of the CaMV 35S promoter ofthe chimeric plasmid pCM 2106 to form the chimeric plasmid pCM 2107 (seealso FIG. 1). The transient gene expression of chimeric plasmid pCM 2107shows that the act1/exon1 sequence in the non-stimulatory chimericplasmid pCM 2106 once again displays its effect of stimulating by atleast a factor of 10 if the plasmid contains the OTF binding site.

It can be seen from the examples of chimeric plasmids pCM 1106 and pCM1107 that the distance to the transcription start site of theheterologous promoter is also very important. Deleting 11 base pairs inthe polylinker of pCM 1106 which are located between the transcriptionstart site and the act1/exon1 sequences abolishes the stimulatory effectof the promoter construct (pCM 1107). Accordingly, the distance betweenthe transcription start site and the act1/exon1 sequence shouldpreferably be at least 11 bp.

The relative values of the increases in gene expression, obtained withthe chimeric plasmids shown in FIG. 5, are given in Table 3 below. Inthis table, the CAT activities of chimeric plasmids pCM 1106, pCM 1107,pCM 2106 and pCM 2107 in Hordeum vulgare protoplasts are related to thatof the chimeric reference plasmid pRT 101 CAT (activity=1).

                  TABLE 3    ______________________________________    pRT 101 CAT              pCM 1106 pCM 1107   pCM 2106                                         pCM 2107    ______________________________________    1         8.1-12.4 0.8-1.2    0.8-1.15                                         8.9-14.5    ______________________________________

The novel modular promoter construct is thus an effective promoter whichis constructed from optionally selected individual regulatory DNAsequences, using which promoter it is possible to increase theexpression of foreign genes in plant cells by a factor of up to at least4000. A DNA sequence from the 5'-non-translated region of exon 1 of therice actin 1 gene is of central importance. This sequence is a ciselement which stimulates RNA polymerase II, which must be locateddownstream of the transcription start site and which only displays itsstimulatory effect on gene expression in the presence of upstreamregulatory DNA sequences.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 1    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ACCACCACCACCACCACCTCCTCCCCCCTCGCTGCCGGACGACGAGCTCCTCCC54    __________________________________________________________________________

We claim:
 1. A modular promoter construct, comprising a promoter whichis active in plant cells and a DNA sequence of at least 30 bases fromexon 1 of the rice actin 1 gene, or derivatives of this modular promoterconstruct which have promoter activity, wherein said promoter is not arice actin 1 gene promoter.
 2. The modular promoter construct accordingto claim 1, wherein said DNA sequence has the sequence of SEQ ID NO: 1.3. The modular promoter construct according to claim 1, furthercomprising a DNA sequence from intron 1 of the maize sucrose synthasegene.
 4. The modular promoter construct according to claim 3, furthercomprising an 18-bp OTF binding site.
 5. A vector comprising a promoterconstruct which comprises a promoter which is active in plant cells anda DNA sequence of at least 30 bases from exon 1 of the rice actin 1gene, or derivatives of this modular promoter construct which havepromoter activity, wherein said promoter construct is coupled to a genewhich is expressed in a plant cell, and wherein said promoter is not arice actin 1 gene promoter.
 6. A plant cell which is transformed with avector comprising a promoter construct which comprises a promoter whichis active in plant cells and a DNA sequence of at least 30 bases fromexon 1 of the rice actin 1 gene, or derivatives of this modular promoterconstruct which have promoter activity, wherein said promoter constructis coupled to a gene which is to be expressed in a plant cell, andwherein said promoter is not a rice actin 1 gene promoter.
 7. A plant orits descendants, regenerated from a plant cell comprising a promoterconstruct which comprises a promoter which is active in plant cells anda DNA sequence of at least 30 bases from exon 1 of the rice actin 1gene, or derivatives of this modular promoter construct which havepromoter activity, wherein said promoter construct is coupled to a genewhich is to be expressed in a plant cell, and wherein said promoter isnot a rice actin 1 gene promoter.
 8. A method for preparing plantshaving elevated gene expression, said method comprising transforming aplant cell with a vector comprising a promoter construct which comprisesa promoter which is active in plant cells and a DNA sequence of at least30 bases from exon 1 of the rice actin 1 gene, or derivatives of thismodular promoter construct which have promoter activity, wherein saidpromoter construct is coupled to a gene which is to be expressed in aplant cell, and wherein said promoter is not a rice actin 1 genepromoter.